The long-term objectives of this proposal are twofold: (1) to identify neurotransmitters released from taste bud cells during gustatory stimulation and (2) to investigate signal processing within the taste bud. These both are significant unanswered questions in the field of chemosensory reception. To date, there is only indirect evidence for candidate neurotransmitters in taste cells, including serotonin, glutamate, norepinephrine, acetylcholine, ATP, and peptides. We have developed a novel methodology to identify the neurotransmitter(s) released from mouse taste buds during gustatory stimulation. Our methodology uses Chinese Hamster Ovary (CHO) cells stably expressing high affinity receptors for candidate neurotransmitters and loaded with the calcium-sensitive dye, Fura 2. The specific aims include investigating whether taste stimulation elicits release of serotonin, norepinephrine, acetylcholine, glutamate, ATP, CCK, and VIP from taste buds. Further, we aim to investigate whether such release is mediated by Ca2+ influx, intracellular Ca2* release, or via transporter mediated mechanisms. We also will study whether gap junctions mediate signal transfer between taste cells during taste stimulation. The project will illuminate information processing in peripheral taste organs and may solve the conundrum of if/how signals generated in one cell (receptor cells) are transmitted to other cells (output cells) in the taste bud. The results may resolve the dilemma that taste cells appear to express a limited set of taste receptors (i.e. bitter, or sweet GPCRs, but not both) yet some taste cells and many sensory afferent nerve fibers respond to multiple taste stimuli: signals generated in dedicated receptor cells are transmitted to other (output) cells and integrated within processing units prior to the final output from the taste bud. [unreadable] [unreadable] [unreadable]